Methylated N-(4-N,N-dimethylaminobenzyl) chitosan for novel effective gene carriers

The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM(47)-Bz(42)-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM(43)-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM(47)-Bz(42)-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM(47)-Bz(42)-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM(43)-CS/DNA and CS/DNA were formed at a ratio of above 1. TM(47)-Bz(42)-CS showed superior transfection efficiency to TM(43)-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM(47)-Bz(42)-CS and TM(43)-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM(47)-Bz(42)-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.     

Incorporation of camptothecin into N-phthaloyl chitosan-g-mPEG self-assembly micellar system.

The capability of N-phthaloylchitosan-grafted poly (ethylene glycol) methyl ether (mPEG)(PLC-g-mPEG) to enhance the aqueous solubility and stability of the lactone form of camptothecin (CPT) was investigated. PLC-g-mPEG formed a core-shell micellar structure after dialysis of the polymer solutions in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) against water, with a critical micelle concentration (CMC) of 28 microg/ml. CPT was loaded into the inner core of the micelles by dialysis method. The results showed an increase in the CPT-loading amount with an increasing concentration of CPT. The stability of drug-loaded micelles was studied by gel-permeation chromatography (GPC), and their in vitro release behaviors were analyzed. Release of CPT from the micelles was sustained. When compared to the unprotected CPT, CPT-loaded PLC-g-mPEG micelles were able to prevent the hydrolysis of the lactone group of the drug. The kinetics of the CPT hydrolysis in human serum albumin (HSA) and fetal bovine serum (FBS) were pseudo-first order. The hydrolysis rate constants for CPT and CPT-loaded PLC-g-mPEG micelles in phosphate-buffered saline (PBS) pH 7.4, were 7.4 x 10(-3) min(-1) and 9.1 x 10(-3) h(-1), parallel to an increase in half-life of CPT from 94 min to 76.15 h, respectively.     

Persistent post-occupational dermatitis

Wall and Gebauer (Contact Dermatitis 1991: 24: 241-243) first described persistent post-occupational dermatitis (PPOD) as ongoing dermatitis for which there is no obvious present cause, precipitated by prior occupational contact dermatitis (OCD). We propose that individuals exhibiting PPOD lose the capacity for resolution of their condition upon removal from exposure to causative agents and subsequently develop persistent dermatitis, which can be continual or intermittent. Accordingly, we suggest modification of criterion 6 of the OCD criteria developed by Mathias (J Am Acad Dermatol 1989: 20: 842-848): 'Removal from exposure initially leads to improvement of dermatitis, however, over time there may be incomplete or no improvement, despite removal from exposures at work'. To satisfy the definition of PPOD, individuals must meet at least 4 of the 7 criteria, including the altered criterion 6. We present 6 cases of PPOD exemplifying these scenarios, which met the altered Mathias criteria. In some cases, subsequent failure to recognize the initial work relatedness of their skin conditions resulted in the termination of workers' compensation benefits. This situation is particularly relevant in the Australian context. The diagnosis of PPOD needs to be considered in all individuals with work-initiated dermatitis who present with ongoing endogenous-like eczema.     

Polymer design and incorporation methods for polymeric micelle carrier system containing water-insoluble anti-cancer agent camptothecin

A water-insoluble anti-cancer agent, camptothecin (CPT) was incorporated to a polymeric micelle carrier system forming from poly(ethylene glycol)-poly(aspartate) block copolymers. Incorporation efficiency and stability were analyzed in correlation with chemical structures of the inner core-forming hydrophobic blocks as well as with incorporation methods. Among three incorporation methods (dialysis, emulsion and evaporation methods), an evaporation method brought about much higher CPT yields with less aggregation than the other two methods. By the evaporation method, CPT was incorporated to polymeric micelles in considerably high yields and with high stability using block copolymers possessing high contents of benzyl and methylnaphtyl ester groups as hydrophobic moieties. This indicates importance of molecular design of the hydrophobic block chain to obtain targeting using polymeric micelle carriers as well as importance of the drug incorporation method.     

Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles.

A water-insoluble antitumor agent, camptothecin (CPT) was successfully incorporated into polymeric micelles formed from poly(ethylene glycol)-poly(benzyl aspartate) block copolymers (CPT-loaded polymeric micelles). Antitumor effects and biodistribution of CPT-loaded micelles were evaluated in mice subcutaneously transplanted by colon 26 tumor cells. Tumor growth was significantly inhibited after a single i.v. injection of CPT-loaded polymeric micelles at doses of either 15 or 30 mg/kg. Efficacy of a single high-dose injection was comparable to low dose multiple injections. CPT loaded in polymeric micelles showed prolonged blood circulation and higher accumulation in tumors compared with CPT in solution. Polymeric micelle systems offer a stable and effective platform for cancer chemotherapy with CPT.     

Physicochemical properties of lipid emulsions formulated with high-load all-trans-retinoic acid

The objective of this study was to formulate high loading with good stability of all-trans retinoic acid (ATRA) lipid emulsion. Lipid emulsions loaded with ATRA were composed of lecithin, medium chain triglyceride and poloxamer-188 or polysorbate-80. The formulation factors in a particular type and amount of oil, emulsifier, and co-emulsifier on the physicochemical properties (i.e., particle size, size distribution, droplets surface charge, pH, percentage yield, drug release, and stability of lipid emulsions) were studied. The particle size of ATRA-loaded lipid emulsions was in the nano-size range of 124.4-378.2 nm with the narrow polydispersity index of 0.04-0.09, which decreased as the amount of co-emulsifiers was increased. The amount of ATRA released from lipid emulsions was operated using a dialysis bag. The receptor medium was ethanol:polysorbate-80:water (10:15:75), adjusted to pH 8.5. ATRA release kinetics in this study were found to follow zero-order kinetics. As the concentration of co-emulsifiers increased, the flux of ATRA released from the lipid emulsions increased. In stability studies, the higher the amount of co-emulsifiers added, the lower the crystallization of ATRA was found. The percentage yield of ATRA was retained at about 70-90% and 60-72% after storage for 60 days at 4 degrees C and 25 degrees C, respectively. These results show a successful incorporation of ATRA into lipid emulsions with high loading capacity and good stability.     

Effect of salt forms and molecular weight of chitosans on in vitro permeability enhancement in intestinal epithelial cells (Caco-2)

The purpose of this study was to investigate the effect of molecular weight (MW) and salt forms of chitosans (aspartate; CS A, glutamate; CS G, lactate; CS L and hydrochloride, CS HCl) on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound for paracellular tight junction transport. Chitosan salts were prepared by spray-drying method. FTIR and solid-state (13)C NMR spectra showed the functional groups of salts in their molecular structures. Salt form, MW of chitosan, and amount of chitosan influenced the permeation-enhancing effects. These studies showed that chitosan salts appeared to increase cell permeability in a dose-dependent manner and caused relatively reversible effects only at the lower doses of 0.001-0.01% w/v. As the MW of chitosan increased from 20 to 460 kDa, the reduction in TEER significantly decreased in the following order: 20 < 45 < 200 < 460 kDa, observed in CS L and CS HCl. In CS A and CS G, the decrease in TEER was not significantly different in all MW because both chitosan salts showed rapid reduction in TEER within 20 min after the start of the experiment. Among chitosan salts, CS A was the most potent absorption enhancer in acidic (pH 6.2) environment. Cytotoxicity of chitosan salts was concentration dependent and varied slightly among the salt forms of chitosan used. CS HCl (MW 45 kDa) was the most toxic having an IC50 of 0.22 +/- 0.06 mg/mL. The ranking of chitosan salts cytotoxicity was CS HCl > CS L> CS G > CS A.     

Methylated N-(4-N,N-dimethylaminocinnamyl) chitosan-coated electrospray OVA-loaded microparticles for oral vaccination

The purpose of this study was to prepare microparticles entrapping ovalbumin (OVA) as a model antigen to induce immune responses in mice following oral vaccination. In this study, calcium-alginate and calcium-yam-alginate microparticles were prepared by crosslinking alginate with calcium chloride solution using an electrospraying technique. 0.1% (w/v) of methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM₆₅CM₅₀CS) was used to coat microparticles entrapping an initial OVA of 20% w/w to polymer. The results indicated that the coated microparticles were spherical and had a smooth surface, with an average size of 1–3 μm, and were positively charged. In addition, the particles demonstrated a greater swelling and mucoadhesive properties than did uncoated microparticles. The in vitro release from the microparticles indicated that the coated microparticles resulted in more sustained release than uncoated microparticles. The cytotoxicity results showed that all of the formulations were safe. The in vivo oral administration demonstrated that at the same amount of 250 μg OVA, coated microparticles exhibited the highest in vivo adjuvant activity in both IgG and IgA immunogenicity.